Epithelial growth factor receptor (EGFR) is a transmembrane protein that is implicated in the progression of many epithelial cancer types. Indeed, several human cancers including, but not limited to, non-small cell lung cancer, squamous cell carcinoma of head and neck cancer, esophageal and gastric cancer, colon cancer, pancreatic cancer, breast cancer, ovarian cancer, bladder cancer, vulvar squamous carcinoma, human androgen-insensitive prostate cancer, renal carcinoma, glioma and glioblastoma displaying EGFR RNA and/or protein overexpression.
EGFR is one of the four homologous transmembrane ErbB proteins that mediate actions of a family of growth factors including EGF, transforming growth factor-α, and the neuregulins. More specifically, EGFR regulates the intracellular effects of ligands such as EGF and TGF-α. Binding of ligands to the EGFR extracellular domains (collectively called the ectodomain) results in allosteric transitions leading to receptor dimerization, protein kinase activation, trans-autophosphorylation, and initiation of signaling cascades (Yarden et al., 2001). The EGFR also interacts with its three known homologues, ErbB2 (HER2), ErbB3 (HER3) and ErbB4 (HER4) in a ligand-dependent fashion to form heterodimers. Heterodimerization of two different members of the ErbB family increases the diversity of ligands recognized by individual receptors and results in an expansion in the repertoire of signaling pathways that can be activated by a given receptor (Jorissen et al., 2003; Olayloye et al., 2000).
Activation of the EGFR induces several transduction pathways inside the cell and contributes to many cellular processes such as cell proliferation, inhibition of apoptosis and angiogenesis. Apoptosis and its underlying pro-apoptotic signaling pathways are often decreased in cancer cells (Zhivotovsky and Orrenius, 2003). Interaction of EGF with its receptor EGFR activates cell proliferation and also blocks death signals (Navolanic et al., 2003). At least in some cases, NF-κB-dependent up-regulation of proliferative and anti-apoptotic genes is responsible for increased cell survival and tumorigenesis (Aggarwal, 2004). In unstimulated cells, NF-κB is usually kept inactive in the cytoplasm through association with inhibitory proteins of the IκB (Inhibitor of NF-κB) family. In response to several stimuli, including pro-inflammatory cytokines such as tumor necrosis factor (TNF) and interleukin-1 (IL-1), IκBα is phosphorylated at serines 32 and 36 by the activity of the IκB kinase (IKK) complex, ubiquitinated and degraded by the proteasome. This allows NF-κB to enter the nucleus, where it is further regulated by phosphorylation, acetylation and interactions with co-activators and co-repressors to transcribe both anti-apoptotic and proliferative genes. It has previously been reported that EGF also induces NF-κB nuclear levels in cell types such as A431 cells and in several breast cancer cell lines that overexpress EGF receptors (Biswas et al., 2003). However, the regulation of NF-κB activity by growth factors such as EGF is less well understood compared to the well-known NF-κB pathway that is activated by TNF. In carcinoma cells that overexpress EGF receptor family members, EGF has been shown to induce IκBα degradation and NF-κB DNA binding (Sun and Carpenter, 1998; Biswas et al., 2000). Likewise, it has been shown that heregulin induces an IKK-dependent, NF-κB-mediated proliferation of estrogen receptor negative, ErbB2 overexpressing breast cancer cells (Biswas et al., 2004) and potentiates ErbB3-mediated NF-κB activation (Bhat-Nakshatri et al., 2002). In addition, up-regulation of IKKα and IKKβ by the integrin-linked kinase/Akt pathway is required for the ErbB2-mediated NF-κB anti-apoptotic pathway (Makino et al., 2004). Additionally, NF-κB-inducing kinase (NIK) has been reported to be complexed with the EGF receptor, which potentiates EGF activation of NF-κB (Chen et al., 2003). Moreover, NIK was shown to potentiate ErbB2/ErbB4-induced NF-κB activation (Chen et al., 2003).
Consistent with EGF-controlled activation of NF-κB, two recent reports have shown positive regulation of the c-fos gene by EGF in quiescent fibroblasts (Anest et al., 2004) and the EAAT2 glutamate transporter gene in astroglioma cells (Sitcheran et al., 2005), through a mechanism involving constitutive nuclear localization of NF-κB. In the latter two cases, EGF-induced NF-κB activation was independent of signaling to IκB. Clearly, further studies are necessary to understand the regulation of EGF-responsive genes by NF-κB.
Due to its role in tumor growth and proliferation, EGFR has been a preferred target for the development of anti-cancer drugs. A first class of anti-EGFR drugs consists of preferably humanized monoclonal antibodies against the extracellular domain of the receptor. Such antibodies have, amongst others, been disclosed in WO 89/06692 and in U.S. Pat. No. 5,470,571. A second class of inhibitors are small molecules that compete with ATP for binding to the ATP site in the EGFR tyrosine kinase domain and, therefore, block the signaling cascade. Gefitinib (ZD1839, Iressa®) is an example of this class. Although these compounds are available, there is still a need for other products that can block EGFR-dependent tumor formation.
Surprisingly, we found that ABIN is also capable of blocking EGF-EGFR-induced cell proliferation. ABIN-1, ABIN-2, and ABIN-3 are three proteins that have been described as inhibitors of TNF, IL-1 and LPS-mediated activation of NF-κB (Heyninck et al., 1999; Van Huffel et al., 2001; Genbank AJ320534). In addition, NF-κB activation mediated by overexpression of the signaling proteins TRADD, RIP, TRAF2 or TRAF6 can be attenuated by co-expression of the ABINs. However, the ABINs have no effect on NF-κB activation induced by overexpression of NIK, IKKβ or the p65 NF-κB subunit. These results indicate that the ABINs act upstream of the IKK complex. Since signaling upstream of IKK is receptor- and stimulus-dependent, the inhibitory effect of ABINs is most likely not applicable to all cases of NF-κB activation, but limited to well-defined pathways. Up to now, there was no indication that ABIN could block ErbB and, more specifically, the EGFR-dependent NF-κB activation, and subsequent EGF-EGFR-dependent proliferation.
A first aspect of the invention is the use of ABIN or an ABIN derivative, or a functional fragment thereof, for the preparation of a medicament to treat an ErbB overexpressing tumor. An ErbB overexpressing tumor means that the tumor tissue shows a higher expressing level of the ErbB member than the same healthy tissue. Preferably, the ErbB overexpressing tumor is selected from the group consisting of EGFR overexpressing tumors and ErbB2 overexpressing tumors. Even more preferably, the ErbB overexpressing tumor is an EGFR overexpressing tumor. The ABIN protein family is known to the person skilled in the art and includes ABIN-1, ABIN-2 and ABIN-3. ABIN and ABIN derivatives as used herein include both nucleic acid, encoding ABIN protein, and the protein itself. Derivatives, as used herein include biologically active mutants and variants of ABIN, and fusion proteins comprising ABIN or a biological active mutant or variant. One preferred embodiment of a derivative is a fusion protein of ABIN with a peptide that promotes delivery of the fusion protein into the cell, such as TAT-derived peptides. Another preferred embodiment of a derivative is a fusion protein of ABIN with a nanobody that can direct the fusion protein to tumor cells. A functional fragment of ABIN or an ABIN derivative is a fragment comprising at least the minimal active domain (MAD). Preferably, the functional fragment consists of the MAD. The MAD as used herein is the minimal domain that still exerts its inhibition on TNF-induced NF-κB activation (Heyninck et al., 2003). The MAD of human ABIN-1 consists of aa 431-588 of human ABIN-1 (accession number AAG42154). On the base of sequence comparison, the MAD of ABIN 2 may be defined as aa 274-429 of ABIN-2 (accession number CAC34835) and the MAD of human ABIN-3 as aa 174-325 of ABIN-3 (accession number AAL02151). The MAD of mouse ABIN-1 consists of aa 444-601 of mouse ABIN-1 (accession number CAB44240) and the MAD of mouse ABIN-2 consists of aa 286-430 of mouse ABIN-2 (accession number CAC34841). Preferably, the functional fragment comprises the MAD of human ABIN-1, more preferably, the functional fragment consists of the MAD of human ABIN-1. The functional fragments may be used on its own, or in a fusion protein as described above.
Nucleic acids encoding ABIN or ABIN derivatives, or functional fragments thereof, can be used in gene therapy. Suitable vectors are known to the person skilled in the art. ABIN and ABIN-derived proteins may be used for direct delivery into the tumor cells. Methods for delivery to tumor cells are known to the person skilled in the art and include, but are not limited to, coupling the protein to tumor-specific antibodies or the use of tumor-specific immunoliposomes. ErbB overexpressing tumors, especially EGFR overexpressing, ErbB2 overexpressing and ErbB3 overexpressing tumors, are known to the person skilled in the art and include, but are not limited to, non-small cell lung cancer, squamous cell carcinoma of head and neck cancer, esophageal and gastric cancer, colon cancer, pancreatic cancer, breast cancer, ovarian cancer, bladder cancer, vulvar squamous carcinoma, human androgen-insensitive prostate cancer, renal carcinoma, glioma and glioblastoma. Preferably, the ErbB overexpressing tumor is squamous carcinoma or human androgen-insensitive prostate cancer.
Another aspect of the invention is the use of ABIN or an ABIN derivative, or a functional fragment thereof, to block EGF-EGFR-dependent cell proliferation.
Still another aspect of the invention is the use of ABIN or an ABIN derivative, or a functional fragment thereof, to inhibit ErbB-dependent NF-κB activation. Preferably, ErbB-dependent NF-κB activation is EGRF-dependent NF-κB activation.